Identification of a mammalian palmitoyltransferase that modifies transmembrane SNARE proteins

SABRINA CHUMPEN RAMIREZ, ALEJANDRA TRENCHI, JAVIER VALDEZ TAUBAS

Punta Ballena

Simposio; Symposium Yeast systems Biology; 2011

Institut Pasteur de Montevideo

S-acylation of proteins is a widespread post-translational modification that consists in the attachment of a lipid moiety to a specific cysteine residue trough a thioester bond. The palmitoyltransferases (PATs), involved in this modification are members of the Asp-His-His-Cys- Cisteine-Rich-Domain (DHHC-CRD) family. Seven members of this family have been identified in Sacharomyces cerevisiae and 23 in mammals. Some members of the SNARE family of membrane fusion proteins contain Transmembrane Domains (TMDs) with adjacent cysteine residues that are palmitoylated both in yeast and in mammals. In S. cerevisiae, this modification is carried out by the Swf1 protein. The mammalian orthologue of Swf1 has not been identified. Although Palmitoyltransferases display very low conservation outside the DHHC domain, sequence analyses point to DHHC4 as a putative Swf1 orthologue. To determine if DHHC4 is the enzyme responsible for mammalian SNAREs palmitoylation we generated a shRNA against the DHHC4 sequence, to knock down the protein expression levels. The efficiency of the knock down was assessed using specific antibodies against the DHHC4 C- terminus, generated in our laboratory. We silenced DHHC4 expression in HEK293 cells and assessed the palmitoylation status of the yeast SNARE Tlg1. We found that Tlg1 palmitoylation is reduced in these cells, suggesting that DHHC4 has palmitoyltransferase activity and might be the mammalian SNARE palmitoyltransferase. Several mammalian SNAREs are currently being tested in the same manner. Additionally, the activity of DHHC4 is also being tested in vitro using purified SNAREs as substrates. It should be noted that the Swf1 orthologue might be responsible for the palmitoylation of other transmembrane proteins with yuxtamembrane cysteines such as glycosyltranferases, viral fusion proteins and BACE 1 protease.